Platelet activation plays an essential role in hemostasis, and dysregulated platelet activation results in arterial thrombosis. Platelet activation is a complex process. Activation causes shape change, granule release, and activation of the major platelet fibrinogen receptor aIIb3. In a subset of activated platelets, additional responses occur that result in drastic changes in the platelet phenotype. Phosphatidylserine is externalized to the surface; the morphology becomes vesiculated and balloon-like; high-levels of 1-granule proteins accumulate on the platelet surface; and additional antigenic and functional changes occur in aIIb3 that result in down-regulation of its activity. All of these events occur subsequent to initial activation responses, such as granule release and shape change, and they are coordinately regulated. Names given to this subset of activated platelets include COAT platelets, coated platelets, SCIP platelets, and highly-activated platelets. Highly-activated platelets have been proposed as regulators of thrombus formation and platelet-neutrophil interactions. However, the physiologic function of highly-activated platelets and the intracellular signaling mechanisms that regulate highly-activated platelet formation remain unclear. A better understanding of the highly-activated platelet's role in thrombus formation may lead to the development of novel hemostatic and anti-thrombotic therapies. Our preliminary studies have identified a critical role for a mitochondrial signaling mechanism in the regulation of highly activated platelet formation. The mitochondrial protein cyclophilin D (CypD) is an essential regulator of mitochondrial permeability transition pore (mPTP) formation. In homozygous CypD-deficient (CypD -/-) mice, mPTP formation in platelets is inhibited, and the transition of platelets from an activated to a highly activated state is almost completely abrogated. The objectives of this application are to determine the physiologic role of platelet mPTP formation and to identify the mechanisms that regulate mPTP formation in platelets. The central hypothesis of this application is that platelet activation by a strong stimulus triggers upstream platelet signaling pathways that result in mPTP and highly-activated platelet formation, and that mPTP and highly activated platelet formation regulate thrombus formation and neutrophil recruitment to the formed thrombus. This hypothesis will be tested by pursuit of the following two interrelated specific aims: 1) Using CypD -/- mice and mice with platelet-specific deletion of CypD, determine the physiologic role of platelet mPTP formation and highly-activated platelet formation in the regulation of thrombosis and neutrophil recruitment, and 2) Identify signaling mechanisms that regulate platelet mPTP formation and highly activated platelet formation. PUBLIC HEALTH RELEVANCE: Dysregulated platelet activation results in arterial clots and thrombotic events like heart disease and stroke. Events within platelet mitochondria regulate formation of an activated platelet subpopulation with unique characteristics that we call highly-activated platelets. Our current study evaluates the role of highly-activated platelets in the regulation of clotting and inflammation using a mouse model with markedly impaired highly- activated platelet formation and examines novel mechanisms that may regulate highly-activated platelet formation.